Selenium, a dietary-antioxidant with cardioprotective effects, prevents the impairments in heart rate and systolic blood pressure in adolescent rats exposed to binge drinking treatment

1 This is an Accepted Manuscript of an article published by Taylor & Francis in Am J 1 Drug Alcohol Abuse on 28/09/2021 available 2 at: https://doi.org/10.1080/00952990.2021.1973485. 3 4 5 Selenium, a dietary-antioxidant with cardioprotective effects, prevents 6 the impairments in heart rate and systolic blood pressure in adolescent 7 rats exposed to binge exposure. 8 Mª Luisa Ojedaa, Paula Sobrinoa, Rui Manuel Ruab, María del Carmen Gallego-Lopez a, Fátima 9 Nogalesa*, Olimpia Carrerasa. 10 aDepartment of Physiology, Faculty of Pharmacy, Seville University, 41012 Seville, Spain. 11 bFaculty of Health Sciences, University Fernando Pessoa, Porto, Portugal. 12 13 *Address: Dra. Fátima Nogales Bueno. 14 Department of Physiology. 15 Faculty of Pharmacy, Seville University. 16 C/ Profesor García González, nº 2. 17 41012. Seville. Spain. 18 Tel: +34 954556518 19 Fax: +34 954233765 20 E-mail: fnogale[email protected] 21 22 Short Title: Cardioprotection by Selenium after adolescent binge exposure. 23 24 2 Word count: 5654 1 2 3 4 Financial Disclosures: The authors report no relevant disclosures 5 Funding: This study was funded by Andalusian Regional Government in its support of CTS-193 6 research group. We also acknowledge the support of the Plan Propio from the University of 7 Seville to promote research and transference activities. 8 9 10 11 12 13 14 15 16 17 18 19 3 1 2 3 4 Abstract 5 Background: Binge drinking (BD) during adolescence has recently been related to 6 cardiovascular alterations. Selenium (Se) is an essential trace element with antioxidant, anti7 inflammatory and antiapoptotic properties, essential for correct heart function. 8 Objectives: To study the protective cardiovascular effects of selenium in adolescent rats 9 exposed to a BD-like procedure. 10 Methods: 32 adolescent male rats exposed to an intraperitoneally BD-like model or not, and 11 supplemented with 0.4ppm of selenite or not, were divided into 4 groups: control, alcohol, 12 control-selenium and alcohol-selenium. At the end of the experimental period blood pressure 13 and heart rate (HR) were determined. Se deposits, oxidative balance and the expression of 14 glutathione peroxidases (GPxs), NF-kB and caspase-3 were measured in the heart. Also, DNA 15 instability in rat lymphocytes and serum vascular markers were determined. Statistical analysis 16 was performed with the ANOVA model. 17 Results: The BD-like model depleted Se heart deposits, decreased GPx activity and GPx1 and 18 GPx4 expression, increased NF-kB, caspase-3 expression, and generated oxidation in myocytes. 19 Outside the heart, BD-like model caused double-strand breaks in lymphocyte DNA and 20 increased the vascular markers Vascular Endothelial Growth Factor (VEGF), Connective Tissue 21 Growth Factor (CTGF). All of these cardiovascular alterations were related to higher systolic 22 and diastolic blood pressure as well as HR. In the heart, Se supplementation of BD-exposed 23 rats significantly increased Se deposits and improved oxidative balance and vascular damage; 24 4 including increased GPxs and decreased NF-kB and caspase-3 activation, consequently 1 decreasing systolic blood pressure and HR. 2 Conclusions: Se-supplementation lowered HR and systolic blood pressure in BD-exposed 3 adolescent rats. 4 Keywords: Binge Drinking Experience; Systolic Blood Pressure; Heart Rate; Selenium; Vascular 5 Markers. 6 1. INTRODUCTION. 7 Repeated binge drinking (BD) is currently the most widespread pattern of alcohol consumption 8 during adolescence [1], yet its effects on the cardiovascular system are poorly understood [2]. 9 Recent clinical studies have shown that this pattern of ethanol consumption in adolescents is 10 associated with a higher risk for hypertension [3] and vascular dysfunction [2]. It has also been 11 related to cardiac arrhythmias in animals [4]. 12 Epidemiological studies report a J-shaped curve relationship between the amount of alcohol 13 consumed and cardiovascular disease [5]. The dose and pattern of alcohol consumption 14 appears to be one of the greatest modulators. During binge drinking episodes blood alcohol 15 levels exceed 0.08% and the risk of adverse alcohol-related consequences increases 16 significantly [6]. 17 It is known that in adults acute ethanol-induced heart alterations are related mainly to its 18 oxidative metabolism; i.e., acetaldehyde and aldehyde dehydrogenases. More than one 19 mechanism is often activated, which leads to a multiple changes in cellular proteins and 20 associated cell functions. For instance, Piano and Phillips [7] revealed important roles for 21 oxidative stress (OS) and the hormone angiotensin II. In this context, it is important to 22 remember that acute ethanol administration during the above indicated oxidative metabolism 23 induces cytochrome P450 2E1 (CYP2E1) activity. Noteworthy is the fact that CYP2E1 is a 24 powerful generator of reactive oxygen species (ROS) [8]. Excessive oxidation can, moreover, 25 5 activate the sympathetic nervous system (SNS) as well as renin-angiotensin-aldosterone 1 system (RAAS) [9]. BD may also adversely affect lipid profiles and hemostatic/coagulation 2 mechanisms, leading to severe peripheral endothelial dysfunction, which affects coronary 3 arteries and heart function. It is also known that a BD pattern directly affects heart function, 4 since it provokes OS and apoptosis and increases the vulnerability for arrhythmias by altering 5 myocardial electrophysiological properties [10,11]. 6 Selenium (Se) is an essential micronutrient found in grain, meat, seafood, and nuts, as well as 7 in dairy products. Se performs its biological functions via its incorporation into 25 different 8 selenoproteins, such as the antioxidant family GPx or the main Se carrier in the body 9 Selenoprotein P (SelP), which also has antioxidant properties [12]. Se, therefore, acts as an 10 antioxidant. Oxidation is related to mitochondrial dysfunction, apoptosis and NF-kB activation, 11 all of which are intimately linked to cardiovascular biology and function. An Se deficiency is, 12 therefore, associated with cardiac pathology [13–16]. 13 In the heart, the most studied selenoprotein is the GPx family [16], by rank the selenoprotein 14 mRNAs expressions detected in the heart are: GPx4 > GPx3 >> GPx1. GPx4 reduces 15 hydroxyperoxides in lipoproteins, complex lipids and phospholipids of biomembranes, and 16 plays an essential antioxidant role in mitochondria, modulating their intrinsic apoptotic 17 pathway and the transcriptional factor NF-KB protein [17]. Due to its ability to serve as an ROS 18 scavenger in extracellular spaces [18], GPx3 protects the extracellular matrix from oxidative 19 damage, while cytosolic GPx1 is particularly important for detoxifying intracellular ROS [14]. 20 SelP is also expressed in the heart; it reduces tissue ROS both directly and also by delivering Se 21 to protect selenoproteins in different tissues[19]. Optimal serum Se levels are, moreover, 22 related to atherosclerosis prevention [20,21]. 23 It has recently been found that BD exposure during adolescence increases ROS and disrupts Se 24 homeostasis and Se tissue distribution [22]. In this context, administering an Se-supplemented 25 6 diet to BD-exposed rats is a good strategy against liver and kidney BD damage, as it decreases 1 oxidative damage and prevents NF-kB activation and apoptosis [23,24]. The objective of this 2 study was to evaluate heart Selenoprotein balance in BD-exposed adolescent rats, and its 3 relationship with oxidative, apoptotic and inflammatory balance, as well as blood pressure, 4 heart rate (HR) and endothelial function. In order to support Se as a therapy against 5 cardiovascular damage in adolescent BD consumers, the above parameters were analyzed 6 after Se supplementation in this animal model. 7 2. MATERIAL AND METHODS. 8 Animals. 9 Thirty-two adolescent male Wistar rats (Centre of Production and Animal 10 experimentation, Vice-rector’s Office for Scientific Research, University of Seville) were used in 11 these experiments. Rats were received at 21 days old and housed in groups of two rats per 12 cage for one week in order to acclimatize them to the housing conditions and handling. The 13 experimental treatment was conducted over a 3–week period, beginning when the rats 14 reached postnatal day (PND) 28 and ending at 47 days of age. This period corresponds to 15 adolescence in Wistar rats [25]. The animals were kept in an automatically controlled 16 temperature (22-23 ºC) vivarium with a 12-hour light-dark cycle (09:00 to 21:00). Animal care 17 procedures and experimental protocols were performed in accordance with EU regulations 18 (Council Directive 86/609/EEC, November 24th 1986) and approved by the Ethics Committee of 19 the University of Seville. 20 On PND 28, when the adolescent period begins, rats were randomly assigned into four groups 21 (n = 8/group) according to their treatments: control group (C): rats were given control diet and 22 drinking water ad libitum, and on the corresponding days, an physiological saline solution (PSS) 23 intraperitoneally (i.p.); binge drinking group (BD): rats were given control diet and drinking 24 water ad libitum, and on the corresponding days, an ethanol solution 20% (v/v) in isotonic 25 saline (3 g/kg/d) i.p.; control selenium supplemented group (CSe): rats were given control diet 26 7 and Se supplemented in drinking water ad libitum, and on the corresponding days an injection 1 of PSS; and binge drinking selenium supplemented group (BDSe): rats were given control diet 2 and drinking water supplemented with Se ad libitum, and on the corresponding days, an 3 alcohol solution 20% (v/v) in isotonic saline (3 g/kg/d) delivered i.p. 4 Standard pellet diet (2014 Teklad Global 14% Protein Rodent Maintenance Diet, Harlan 5 Laboratories, Barcelona, Spain) that contained 0.23 ppm of Se were available ad libitum in all 6 the experimental groups. The Se supplemented groups (CSe and BDSe) received 0.14 ppm of 7 Se of extra as anhydrous sodium selenite (Panreac, Barcelona, Spain) in drinking water during 8 all experimental period. The amount of 0.14 ppm of Se was chosen according to the amount of 9 Se consumed by adolescent rats just from the standard pellet diet, and based on the study of 10 Yang et al. [26], which, states that GPx activities in rats‘ plasma and liver were maximized at 11 0.5 ppm of dietary Se. Since C adolescent rats used in this study presented an intake of 12 approximately 12 g of food per day, with a supplemented Se diet of 0.5 ppm they will receive 6 13 µg/day of Se. With the objective of studying the effects of excessive but not extreme Se doses, 14 the amount of Se supplemented in water was estimated in order not to reach 6 µg/day of Se 15 intake. 16 Nutritional control. 17 Body weight and the amount of food consumed by rats were monitored daily until the end of 18 the experimental period. The amount of food and drinking water ingested was calculated by 19 measuring these parameters every day in the morning. Knowing the Se concentration (ppm) in 20 the diet and the drinking water, Se intake was calculated by multiplying by food and water 21 ingested every day. All measurements were taken at 9:00 a.m. to avoid changes due to 22 circadian rhythms. 23 Ethanol treatment. 24 8 Alcohol-exposed groups (BD and BDSe) received an i.p. injection of alcohol (20 % v/v) in PSS (3 1 g/kg/d). Alcohol injections were given starting at 7:00 p.m., when the dark cycle began, for 3 2 consecutive days each week for 3 weeks. No i.p. injections were given during the remaining 4 3 days of each week [27]. Control groups (C and CSe) received an i.p. injection of an equal 4 volume of PSS at the same time as the alcohol-exposed group’s injections. 5 Blood pressure (mm Hg) and heart rate (beats/min). 6 Systolic and diastolic blood pressure (SBP and DBP), and heart rate (HR) were monitored with a 7 pressure meter (NIPREM 645, CIBERTEC, Spain) using the indirect tail occlusion method. 8 Measurements were taken in adolescent rats 24 hours after last alcohol exposure or last 9 injection with saline solution. Each animal was measured 4-5 times successively in order to 10 calculate the arithmetic mean, this being the value analyzed. Mean blood pressure (MBP) was 11 calculated from the SBP and DBP data. 12 Samples. 13 At the end of the experimental period the rats were fasted for 12 h and feces and urine 14 samples were collected using individual metabolic cages. Then, 24 hours after their last alcohol 15 exposure or treatment with saline solution, adolescent rats were anesthetized with an i.p. 16 injection of 28% w/v urethane (0.5 ml/100 g of body weight). The blood was obtained by heart 17 puncture and collected in tubes. The serum was prepared using low-speed centrifugation for 18 15 min. at 1300 x g. The abdomen was opened by a midline incision and the whole heart was 19 removed, weighed, and frozen in liquid nitrogen and stored at −80 ºC for future biochemical 20 determinations. 21 Selenium Analysis. 22 Serum and heart Se levels were determined by graphite-furnace atomic absorption 23 spectrometry following the procedure described in Ojeda et al. [28]. We used a PerkinElmer 24 AAnalyst™ 800 high-performance atomic absorption spectrometer with WinLab32 for AA 25 9 software, equipped with a Transversely Heated Graphite Furnace (THGA) with longitudinal 1 Zeeman-effect background corrector and an AS-furnace autosampler (PerkinElmer, 2 Ueberlingen, Germany) and electrodeless Se discharge lamps (EDLs). Further details are 3 provided in the supplementary material. 4 Antioxidant enzymes and oxidative stress markers. 5 The activity of antioxidant enzymes (Superoxide dismutase (SOD), catalase (CAT), GPx and 6 glutathione reductase (GR)), as well as lipid and protein oxidation, were determined in the 7 heart, employing the biochemical assays described in Ojeda et al. [29]. Further details are 8 provided in the supplementary material. 9 Immunoblotting assays. 10 The expression of the selenoproteins GPx1, GPx3, GPx4 and SelP as well as NF-κB p65 and 11 cleaved caspase-3 were determined in whole-heart samples of the adolescent rats by Western 12 blotting. The samples contained 150 µg of protein. The specific primary antibodies (rabbit 13 polyclonal IgG, Santa Cruz Biotechnology) were diluted: GPx1 and GPx3 (1:2000), GPx4 14 (1:5000), and SelP (1:2500), NF κB p-65 and cleaved caspase-3 (1:1000). Secondary antibody 15 (anti-rabbit IgG HRP conjugate, Santa Cruz Biotechnology) was utilized in dilutions of 1:5000 16 for GPx1, GPx3 and SelP, 1:10000 for GPx4, 1:2500 for NF κB p-65 and caspase-3. Further 17 details are provided in the supplementary material. 18 Lymphocytes isolation and determination of DNA stability by comet analysis. 19 DNA instability (strand breaks) was measured in rat lymphocytes at the age of 47 days by 20 comet analysis [30]. The percentage of fluorescence in the comet tail, head and Olive Tail 21 Moment (OTM, defined as the product of the tail length and the fraction of total DNA in the 22 tail, OTM = [Tail mean − Head mean] × Tail % DNA/100) were measured. Further details are 23 provided in the supplementary material. 24 Vascular markers. 25 16 As it dramatically increases DNA damage in lymphocytes, BD leads to higher systemic 1 oxidation. It is established that acute ethanol induces DNA damage in peripheral lymphocytes 2 via an oxidative pathway [47]. Therefore, in this study BD increased the OTM of lymphocytes, 3 indicating the breaking of the double helix, one of the most severe types of DNA damage, due 4 to the oxidation generated. Se supplementation completely prevents this. This prevention 5 probably occurs by increasing GPx activity and decreasing OS in these cells. In fact, in other 6 studies, Se supplementation administered to adolescent rats exposed to the same BD protocol 7 as the one used in this study increased serum GPx3 activity and decreased serum lipid 8 peroxidation, both of which were altered in ethanol-exposed animals [24]. These results are 9 interesting, since systemic oxidation and serum GPx3 activity are intimately related to vascular 10 function and blood pressure [48]. These are both conditions which are highly compromised in 11 our BD model, such that a single acute ethanol exposure enhances vascular OS and induces 12 vascular dysfunction through RAAS activation as well as vascular and systemic lipid 13 peroxidation [49]. We have previously found that adolescent BD rats have high serum 14 aldosterone levels and lipid peroxidation [24] and that Se supplementation decreases both 15 parameters, but only modestly. Perhaps this is the reason why the effects of Se upon blood 16 pressure are lower than in heart function. 17 The intermittent BD pattern used in adolescent rats increased all of the vascular markers 18 studied here in. VEGF is considered the most potent proangiogenic growth factor involved in 19 vascular permeability, vascular dilation, endothelial proliferation and angiogenesis. Previous 20 data established that acute ethanol exposure significantly increases serum VEGF values, but 21 also perturbs endothelial VEGF signalling and action [50,51]. The growth factor CTGF plays 22 important roles in cell adhesion, migration, proliferation, and angiogenesis and is critically 23 involved in fibrotic process. Different studies relate CTGF to VEGF production and angiogenesis 24 [52]. CTGF is upregulated by stimuli involved in cardiovascular damage, including OS [53]. Since 25 ethanol exposure increases OS, both proteins (CTGF and VEGF) increased in this study and 26 17 endothelial function is compromised. Se supplementation improves vascular function, 1 lowering SBP values by decreasing both parameters to normal values, probably due to its 2 antioxidant properties. The observation of beneficial actions of Se on vasculature are not new, 3 since anti-atherosclerotic activity of Se has been described previously [21]. 4 tPAI-1 is an acute phase protein expressed in adipocytes and endothelial cells, and is highly 5 expressed by most cells in response to stress. tPAI-1´s role has been identified as the inhibition 6 of plasminogen activator which blocks fibrinolysis. Liver can produce large amounts of tPAI-1 in 7 response to stress, such as ethanol exposure; furthermore, tPAI-1 plays a critical role in 8 alcohol-induced steatosis [49,50]. Therefore, BD exposure can increase this parameter greatly, 9 resulting in vascular and hepatic damage. However, despite the fact that Se supplementation 10 in adolescent BD animals improved hepatic inflammatory, oxidative and apoptotic profile and 11 function [23], although it does not decrease tPAI-1 serum levels. Moreover, tPAI-1 serum levels 12 also increased in control Se-supplemented animals. Although this result was new to our 13 laboratory, Viezeliene et al. [55] found similar results when examining aluminum-induced OS 14 and the effects of supplementing the animals with Se. Se is a mineral related to lipid 15 homeostasis [56]. It is also known that t-PAI-1 secretion in HepG2 cells is modulated by 16 triacylglycerols and by linoleic acid and/or its metabolic products [57]. Therefore, perhaps Se 17 and tPAI-1 synthesis could be related via lipid homeostasis. 18 Cav-1 is the main component of the caveolae plasma membranes found in most cell types and 19 it interacts with different cell components, regulating multiple cellular events such as cell 20 growth, apoptosis and cholesterol trafficking. It allows blood vessels to sense, organise and 21 mediate signal transduction in the face of altered shear stress conditions [58]. The underlying 22 mechanism by which Cav-1 regulates ethanol induced damage is, however, not well23 understood. In a BD model [59], it was found that an increase in serum and hepatic Cav-1, 24 which could be a cellular defense through inhibiting reactive nitrogen species and iNOS25 18 signalling cascades. In our BD model, serum Cav-1 values increased and Se supplementation 1 increased this value even further. Control Se animals also had high Cav-1 serum levels. Thus, Se 2 appears to be related to serum Cav-1 levels, yet there are no bibliographical data which reflect 3 or could explain this effect. We think that since this mineral is related to lipid homeostasis and 4 Cav-1 is also important in cholesterol trafficking and insulin resistance, they could be related at 5 different points in these pathways [60]. 6 In summary, BD exposure during adolescence severely damages cardiac tissue by 7 generating oxidation, inflammation and apoptosis, leading to high HR. These disturbances 8 could be related in part to the low Se and GPx4 levels found in the heart, since they contribute 9 to decrease mitochondrial oxidation, increasing their survival and function. Therefore, Se 10 supplementation appears to be a good strategy for preventing heart oxidation, inflammation 11 and apoptosis, improving heart function by reducing the tachycardia generated by BD 12 exposure. BD-exposed rats also developed high SBP and DBP, due to higher systemic oxidation 13 generated, which leads to endothelial dysfunction. Se supplementation in BD-exposed rats 14 reverses DNA oxidation and the serum levels of the proangiogenic markers. However, these 15 actions result in a partial decrease in SBP only. Public health messages regarding BD should, 16 therefore, include information on the cardiovascular effects of this consumption pattern, since 17 it provokes heart damage and vascular dysfunction, which also are important risk factors 18 during the third and fourth decades of life. In this context, Se therapy should be investigated 19 further as an antioxidant strategy for preventing such cardiovascular damage. 20 Conflict of interest: All authors of this manuscript declare that there are no conflicts of 21 interest. 22 4. REFERENCE. 23 [1] G. Martinotti, M. Lupi, L. Carlucci, R. Santacroce, E. Cinosi, T. Acciavatti, F. Sarchione, V. 24 Verrastro, P. Diotaiuti, I. 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Nutritional and heart parameters. 5 6 C BD CSe BDSe Kcal intake (kcal/day) 54.6 ± 3.4 50.1 ± 3.3 56.9 ± 3.5 50.5 ± 2.9 Increased body weight (g/day) 5.7 ± 0.2 5.1 ± 0.3 5.9 ± 0.2 5.2 ± 0.2 Se intake (µg/day) 3.1 ± 0.2 2.8 ± 0.2 aaa 5.6 ± 0.3 ccc 4.9 ± 0.3 HRW (%) 0.52 ± 0.024 0.52 ± 0.011 0.47 ± 0.009 0.50 ± 0.023 Heart Protein (mg prot/ml) 9.82 ± 0.64 9.03 ± 0.67 8.34 ± 0.50 9.04 ± 0.73 Se in heart (µg Se/g dry weight) 0.12 ± 0.005 0.09 ± 0.01 **, aaa 0.18 ± 0.01 cc 0.17 ± 0.01 7 The results are expressed as mean ± SEM and analysed by a multifactorial analysis of variance (one-way 8 ANOVA) followed by the Tukey´s test. The number of animals in each group is 8. HRW: heart relative 9 weight; Se: Selenium. Groups: C: control group, BD: binge drinking group, CSe: control selenium 10 supplemented group, and BDSe: binge drinking selenium supplemented group. Statistic difference 11 between groups was expressed as: A vs C: **p<0.01; BD vs BDSe: aaap<0.001; C vs CSe: CCp<0.01, 12 CCCp<0.001. 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 25 1 2 3 4 Table 2. Oxidative balance in heart: antioxidant enzymes activities (SOD, CAT, GPx and GR) 5 and lipid (MDA) and protein (PC) oxidation. 6 7 C BD CSe BDSe SOD activity (U/mg proteins) 3.9 ± 0.2 4.3 ± 0.3 3.9 ± 0.1 4.2 ± 0.3 CAT activity (U/mg proteins) 28.1 ± 1.9 48.1 ± 2.7 ***,a 32.7 ± 1.9 37.5 ± 3.2 GPx activity (U/mg proteins) 106.8 ± 4.2 86.3 ± 3.3 **,aaa 169.5 ± 6.1 cc 146.5 ± 7.1 GR activity (U/mg proteins) 7.8 ± 0.6 11.2 ± 0.6 ***,aaa 7.7 ± 0.5 6.1 ± 0.5 MDA (mol/mg proteins) 0.056 ± 0.004 0.249 ± 0.019 ***,aaa 0.056 ± 0.004 0.051 ± 0.004 PC (mol/mg proteins) 5.3 ± 0.1 7.2 ± 0.2 ***,a 5.8 ± 0.2 6.4 ± 0.2 8 The results are expressed as mean ± SEM and analyzed by a multifactorial analysis of variance (one-way 9 ANOVA) followed by the Tukey´s test. The number of animals in each group is 8. SOD: superoxide 10 dismutase; CAT: catalase; GPx: glutathione peroxidase; GR: glutathione reductase; MDA: 11 malondialdehyde; PC: protein carbonyl. Groups: C: control group, BD: binge drinking group, CSe: control 12 selenium supplemented group, and BDSe: binge drinking selenium supplemented group. Statistic 13 difference between groups was expressed as: BD vs C: **p<0.01, ***p<0.001; BD vs BDSe: a p<0.05, aaa 14 p<0.001; C vs CSe: CCp<0.01. 15 16 17 18 19 20 21 22